sexta-feira, outubro 24, 2014

Topoisomerase II Is Required for the Proper Separation of Heterochromatic Regions during Drosophila melanogaster Female Meiosis

Stacie E. Hughes mail, R. Scott Hawley mail

Published: October 23, 2014DOI: 10.1371/journal.pgen.1004650

Abstract

Heterochromatic homology ensures the segregation of achiasmate chromosomes during meiosis I in Drosophila melanogaster females, perhaps as a consequence of the heterochromatic threads that connect achiasmate homologs during prometaphase I. Here, we ask how these threads, and other possible heterochromatic entanglements, are resolved prior to anaphase I. We show that the knockdown of Topoisomerase II (Top2) by RNAi in the later stages of meiosis results in a specific defect in the separation of heterochromatic regions after spindle assembly. In Top2 RNAi-expressing oocytes, heterochromatic regions of both achiasmate and chiasmate chromosomes often failed to separate during prometaphase I and metaphase I. Heterochromatic regions were stretched into long, abnormal projections with centromeres localizing near the tips of the projections in some oocytes. Despite these anomalies, we observed bipolar spindles in most Top2 RNAi-expressing oocytes, although the obligately achiasmate 4th chromosomes exhibited a near complete failure to move toward the spindle poles during prometaphase I. Both achiasmate and chiasmate chromosomes displayed defects in biorientation. Given that euchromatic regions separate much earlier in prophase, no defects were expected or observed in the ability of euchromatic regions to separate during late prophase upon knockdown of Top2 at mid-prophase. Finally, embryos from Top2 RNAi-expressing females frequently failed to initiate mitotic divisions. These data suggest both that Topoisomerase II is involved in the resolution of heterochromatic DNA entanglements during meiosis I and that these entanglements must be resolved in order to complete meiosis.

Author Summary

Proper chromosome segregation during egg and sperm development is crucial to prevent birth defects and miscarriage. During chromosome replication, DNA entanglements are created that must be resolved before chromosomes can fully separate. In the oocytes of the fruit fly Drosophila melanogaster, DNA entanglements persist between heterochromatic regions of the chromosomes until after spindle assembly and may facilitate the proper segregation of chromosomes during meiosis. Topoisomerase II enzymes can resolve DNA entanglements by cutting and untwisting tangled DNA. Decreasing Topoisomerase II (Top2) levels in the ovaries of fruit flies led to sterility. RNAi knockdown of the Top2 gene in oocytes resulted in chromosomes that failed to fully separate their heterochromatic regions during meiosis I and caused oocytes to arrest in meiosis I. These studies demonstrate that the Top2 enzyme is required for releasing DNA entanglements between homologous chromosomes before the onset of chromosome segregation during Drosophila female meiosis.

Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. The Stowers Institute for Medical Reserach has made all primary data files accessible at the following web page: http://odr.stowers.org/websimr/datasetvi​ew/673/0/.

Funding: RSH is supported by Stowers Institute for Medical Research and is an American Cancer Research Professor supported by the award RP-05-086-06DDC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.